Comparative genetic characterization of CMY-2-type beta-lactamase producing pathogenic Escherichia coli isolated from humans and pigs suffering from diarrhea in Korea

Background Pathogenic Escherichia coli are an important cause of bacterial infections in both humans and pigs and many of antimicrobials are used for the treatment of E. coli infection. The objective of this study was to investigate the characteristics and relationship between humans and pigs regarding third-generation cephalosporin resistance and CMY-2-producing E. coli in Korea. Results All 103 third-generation cephalosporin-resistant E. coli isolates showed multidrug resistance. Also, except for β-lactam/β-lactamase inhibitor combinations, all antimicrobials resistant rates were higher in pigs than in humans. A total of 36 isolates (humans: five isolates; pigs: 31 isolates) were positive for the CMY-2-encoding genes and thirty-two (88.9%) isolates detected class 1 integrons with 10 different gene cassette arrangements, and only 1 isolate detected a class 2 integron. The most common virulence genes in pigs were LT (71.0%), F18 (51.6%), and STb (51.6%), while stx2 (80.0%) was the most frequently detected gene in humans. Stx2 gene was also detected in pigs (6.5%). Interestingly, 36 CMY-2-producing E. coli isolates showed a high diversity of sequence types (ST), and ST88 was present in E. coli from both pigs (11 isolates) and humans (one isolate). Conclusion Our findings suggest that a critical need for comprehensive surveillance of third-generation cephalosporin resistance is necessary to preserve the usefulness of third-generation cephalosporins in both humans and pigs. Supplementary Information The online version contains supplementary material available at 10.1186/s12941-023-00559-1.


Introduction
Escherichia coli is member of a large bacterial family, Enterobacteriaceae, which consists of facultative anaerobic Gram-negative rods that live in the intestinal microflora of humans and animals. Although many E. coli are harmless commensals, pathogenic E. coli are an important cause of bacterial infections like colibacillosis. In humans, these strains are the foremost cause of diarrhea and hemorrhagic colitis as well as hemolytic uremic syndrome [1]. In pigs, these strains cause diarrhea with edema disease and economic loss on pig farming due to decreasing weight gain, and costs for feed supplements, vaccinations and treatments [2]. Trimethoprim-sulfamethoxazole, fluoroquinolones, and third-generation cephalosporins are the important antimicrobials for treating infections caused by pathogenic E. coli. Thirdgeneration cephalosporins are widely used for the treatment of bacterial infections in both human and veterinary medicines [3]. Misuse of third-generation cephalosporins increases the emergence of extended-spectrum β-lactamase (ESBL) and AmpC β-lactamases producing E. coli isolates in humans and animals [4][5][6]. These isolates are resistant to β-lactam antibiotics because the ESBL and AmpC β-lactamases allow bacteria to hydrolyze β-lactam antibiotics. AmpC beta-lactamase genes can be located and encoded on chromosomes or plasmids [7].
In pathogenic E. coli, antimicrobial resistant genes and virulence genes are frequently detected in isolates from both humans and pigs. Pigs are considered the primary reservoirs of pathogenic E. coli which can lead the contamination of food products such as pork, and human infection [11][12][13]. Although some studies about thirdgeneration cephalosporins resistance of pathogenic E. coli have been reported in either human or pigs [4,5,8], a relative paucity of information exists showing a relationship between humans and pigs. Therefore, this study aimed to investigate the characteristics and relationship between pathogenic isolates from humans and pigs with third-generation cephalosporin resistance of CMY-2-producing E. coli.

Escherichia coli isolates
Between 2008 and 2020, 392 pathogenic E. coli isolates were collected from 401 pigs with colibacillosis clinical case in 120 different farms, respectively. The farms consisted of different pig herds (50 to 100 sows per each herd). Samples were not collected repeatedly from the same farm. The aseptically collected intestinal contents and feces were inoculated on MacConkey agar (BD Biosciences, Sparks, MD) and Eosin methylene blue agar (BD Biosciences) and incubated at 37℃ for 20 to 24 h. Only one typical colony was selected from each sample and transferred to blood agar. Suspected colonies were identified as E. coli using the VITEK II system (bioMéreiux, Craponne, France). Hemolysis was also determined in blood agar (Asan Pharmaceutical, Seoul, Korea). Also, 197 strains from 197 diarrheic patients from 1981-2019 were provided by the National Culture Collection for Pathogens (NCCP; Korea, 51 strains), Gyeongsang National University Hospital Branch of the NCCP (GNUH-NCCP; Korea, 138 strains), and Kyungpook National University Hospital Branch of the NCCP (KNUH-NCCP; Korea, eight strains).

Third-generation cephalosporin resistant E. coli identification
Two-hundred microliters of standardized inoculum (0.5 McFarland) of each E. coli isolate (collected on MacConkey agar without antimicrobial) was plated on Mueller-Hinton agar plates supplemented with 2 μg /mL cefotaxime (Sigma-Aldrich, St.Louis, MO) and incubated at 37 ℃ for 24 h to select third-generation cephalosporinresistant E. coli [14]. Ultimately, a total of 103 third-generation cephalosporin-resistant E. coli strains (8 strains isolated from KNUH-NCCP, 15 strains isolated from GNUH-NCCP, 8 strains isolated from NCCP, and 72 strains isolated from 19 different pig farms in this study) were tested in this study (Additional file 1).

Detection of β-lactamase-encoding genes
PCR amplification was conducted with primers (Table 1) for CTX-M, TEM, SHV, OXA, and pAmpC β-lactamase genes in the 103 third-generation cephalosporin-resistant E. coli. PCR products were sequenced using an automatic sequencer (Cosmogenetech, Seoul, Korea). The sequences were confirmed with those in the GenBank nucleotide database using the Basic Local Alignment Search Tool (BLAST) program available through the National Center for Biotechnology Information website (http:// www. ncbi. nlm. nih. gov/ BLAST). Therefore, 36 CMY-2-producing E. coli isolates were identified from 103 third-generation cephalosporin-resistant E. coli isolates.

Plasmid replicon typing and detection of integrons and gene cassettes
For plasmid replicon typing and detection of integrons and gene cassettes, PCR amplification was performed using DNA extracted from CMY-2-producing E. coli isolates. The DNA was extracted as described above. The primers used in this study targeted 18 different replicons [32] and class 1 and 2 integrons (Table 1). Gene cassettes were tested for integron-positive isolates. The PCR products of the gene cassettes were sequenced as described above (Additional file 2).

Multi-locus sequence typing (MLST)
All processes, including genomic DNA extraction, PCR amplification, Sanger sequencing, and assembly were performed by Macrogen (Macrogen, Seoul, South Korea). Genomic DNA were extracted using a QIAamp

Statistical and data analysis
The statistical package SPSS 23 was used for the description of antimicrobial patterns. A comparison of antimicrobial resistance rate between third-generation cephalosporin-resistant E. coli isolated from human and third-generation cephalosporin-resistant E. coli isolated from pig was conducted with the two-sample t-test. Differences were considered significant at P < 0.05.

Discussion
In our study, all third-generation cephalosporin-resistant E. coli were identified multidrug-resistant and were nonsusceptible to β-lactam antimicrobials like penicillins. These results indicate that third-generation cephalosporin-resistant E. coli show co-association of resistance to other classes of antimicrobials and high MDR rates. Also, although high resistance frequencies about non-beta-lactam antimicrobials were identified for aminoglycosides (98.6%), phenicols (91.7%), tetracycline (99.9%), quinolone (86.1%), and folate pathway inhibitors (80.6%) in pigs, only aminoglycoside resistance (90.3%) showed high frequency in humans. In animals, antimicrobial agents are used in large amounts to promote animal growth as well as to prevent and treat diseases [33,34]. Therefore, the widespread use and misuse of antimicrobials in animal has resulted in the emergence of antimicrobial-resistant bacteria and it can get transferred to humans. ESBL and pAmpC β-lactamase genes emerge when third-generation cephalosporins are overused and misused for prevention and treatment, which is a common mechanism of resistance to third-generation cephalosporins. In particular, the CMY-2-encoding gene is the most dominant pAmpC β-lactamase gene in E. coli isolates from both human and food producing animals. In Europe, the CMY-2-encoding gene has been detected in third-generation cephalosporin-resistant E. coli isolated in humans as well as in pigs [6,35,36]. In china, the prevalence of CMY-2-producing E. coli has been reported from food and domestic animals [37,38]. Moreover, in Korea, CMY-2-encoding gene have been observed among animal and human E. coli isolates [5,39,40]. In this study, TEM-1 and OXA-1, enzymes conferring β-lactam resistance, were detected in 27 (75.0%) and 4 (11.1%) isolates, respectively. These genes are not ESBL or pAmpC β-lactamases, but can be transformed into ESBL by mutations that alter the amino acid sequence around the active site [41]. The CMY-2 gene can co-exist with other classes of antimicrobials genes in mobile genetic elements, and thus CMY-2-producing E. coli are commonly MDR [7,42]. In this study, all 36 CMY-2-producing E. coli isolates carried a variety of antimicrobial resistance genes including sul1, sul2, catA1, cmlA, aac(6')-Ib, aac(3)-II, tetA, tetB, tetE, and qnrS. The tetB gene was identified in CMY-2-producing E. coli isolates in both pigs and humans, which is consistent with the findings of Koga et al. and Endimiani et al. [7,43]. The sul1 and sul2 genes, which encode sulfonamide-resistant dihydropteroate synthase, were identified in 15 (41.7%) and 27 (75.0%) CMY-2-producing E. coli isolates, respectively, and the aac(3)-II and aac(6')-Ib genes, which encode aminoglycoside adenylyltransferase, were detected in 11 (30.6%) and 7 (19.4%) CMY-2-producing E. coli isolates, respectively. These genes have already been reported as major determinants of sulfonamides (sul1 and sul2) and gentamicin (aac(3)-II and aac(6')-Ib) resistance in Gram-negative bacteria [44]. Although chloramphenicol is banned in food-producing animals because of its suspected carcinogenicity [45], we found that 18 (50.0%) and one (2.8%) of 31 CMY-2-producing E. coli isolated from pigs carried the cmlA and catA1 genes, respectively; these genes encodes a specific chloramphenicol transporter. Also, florfenicol which has been shown to have a spectrum of activity similar to that of chloramphenicol and used in veterinary medicine is related to chloramphenicol and can select for cross-resistance among bacterial pathogens [46,47]. Therefore, although chloramphenicol is already prohibited worldwide in food animals, there is a reservoir of chloramphenicol resistance in bacteria from food animals, which can disseminate on transferable plasmids, remains a concern as chloramphenicol is a useful antibiotic for the treatment of bacterial infections in humans [48,49]. qnrS genes, which encodes plasmid-mediated quinolone resistance, were identified in both pigs (32.3%) and humans (40.0%). Previous studies reported that the PMQR genes in β-lactamases-producing-E. coli were detected at high levels [50]. The presence of PMQR genes in β-lactamases-producing Enterobacteriaceae may be due to common carriage on a plasmid [51].
Integrons are genetic elements that transfer antimicrobial resistance gene, and they play an important role in conferring resistance to multiple antimicrobials [52]. In recent years, integrons have been found in β-lactamaseproducing isolates of Gram-negative bacteria [53,54]. In this study, the aadA gene was the most prevalent gene cassette of the integrons followed by the dfrA gene. These genes are also frequently detected in gene cassettes of integrons isolated from humans and pigs in Korea [55,56]. Because β-lactamase-producing isolates harboring the aadA or dfrA or both genes showed higher antimicrobial resistance rates [57,58], the selection of antimicrobials for the treatment of colibacillosis remains a serious concern. Also, the most common plasmid replicons were IncF plasmids including FIB (83.3%), FIC (52.8%), and FIA (33.3%). IncF plasmids are associated with important role in the worldwide emergence and spread of virulence and antimicrobial resistance determinants including extended-spectrum β-lactamases and pAmpC genes (CMY and DHA) among pathogenic E. coli [59].
For diagnosis and preventative measures for colibacillosis, detection of E. coli virulence factors is important [60]. In this study, LT, STb, F18, and F4 were detected in 22 (71.0%), 16 (51.6%), 16 (51.6%) and 11 (35.3%) CMY-2-producing E. coli isolates from pigs, respectively. The LT gene play a significant role in producing heat-labile enterotoxins and causing diseases [61].ST genes damage vessels and cause edema leading to high mortality in pigs [62]. Further, adhesive fimbriae gene including F4, F5, F6, F18, and F41 play important roles in allowing pathogenic E. coli to attach to the epithelial cells and intestinal mucosa and in causing diseases [58]. Interestingly, inactivated vaccines targeting F4 and F18 are being used in Korea [63]. The use of these vaccines could cause antigenic variations and would account for the prevalence of F4 and F18, in pigs. The stx2 gene was also detected in isolates from both pigs (two isolates, 6.5%) and humans (four isolates, 80.0%). The stx gene is associated with edema disease in swine and hemolytic-uremic syndrome in humans [64][65][66] and the receptor for stx2 is globotriosyl ceramide, which is found in both humans and pigs.
MLST help to identify the phylogenetic relationships among deep lineages, providing a view of the population structure of bacterial species [67]. In this study, we found eleven STs, including eight STs (ST1, ST10, ST88, ST100, ST602, ST641, ST953, and ST1642) from pigs and four STs (ST88, ST131, ST410, and ST1308) from humans. The most prevalent STs in pigs were ST88 (12 isolates), ST100 (seven isolates), and ST10 (six isolates), which are the predominant ETEC type, and are important pig pathogens in the many country (Canada, Germany, Thailand, and United States (http:// mlst. warwi ck. ac. uk/ mlst/ dbs/ Ecoli). In particular, ST88 was reported in both humans and pigs and it has been previously described in association with antimicrobial resistant gene like AmpC [68]. These ST is related to strains pathogenic and antimicrobial resistance and emergence of similar ST might indicates transmission between pigs and humans [69,70].

Conclusions
In this study, we genetically analyzed, characterized, and investigated the prevalence and relationship of third-generation cephalosporin resistance and CMY-2-producing E. coli isolated from humans and pigs in Korea suffering from diarrhea. To our knowledge, this is the first study to investigate the molecular characteristics and relationship between third-generation cephalosporin-resistant and CMY-2-producing E. coli isolated from humans and pigs in Korea. Third-generation cephalosporin resistant bacteria can get transferred to humans through the food chain and lead to treatment failure of serious infections. Therefore, a critical need for comprehensive surveillance of thirdgeneration cephalosporin resistance is necessary to preserve the usefulness of third-generation cephalosporins in both humans and pigs.